Embyoid organizer

Recapitulation of development in vitro relies primarily on treatment of progenitor cells with media-borne morphogens, without the complex spatial instructions normally presented by native developmental niches. Here we build simple spatially asymmetric developmental niches by engineering synthetic organizer cells that self-assemble around stem cells and provide spatially-defined morphogenic instructions. Using a toolkit of natural and engineered adhesion molecules we harness differential adhesion to program the formation of specific organizer cell geometries around the stem cells, allowing the generation of morphogen gradients with tunable position, amplitude, and signal types. Synthetic niches presenting the antagonistic morphogens WNT3A and DKK1 were used to guide development of pluripotent stem cells. These synthetic organizers generated distinct morphological outcomes, including formation of a beating heart-like chamber and embryoids containing a complement of diverse cell lineages derived from mesoderm, endoderm and ectoderm. The resulting morphologies were reproducible in high-throughput format. Spatially programmable synthetic organizer cells, which integrate the principles of differential adhesion and positional information, thus provide a systematic approach to guide, recapitulate, and modify development in a controlled fashion. Embryoids were combined with either a WNT3A-producing node, a DKK1-producing node, or both. WNT3A expression was induced on day 1, DKK1 expression on day 2. Embryoids from 300 mESC were collected on day 8, dissociated, and cells were labelled for MULTIseq then sorted for viability.